Angiogenesis is the fundamental process by which new blood vessels are formed. The process comprises the migration of vascular endothelial cells into a tissue, followed by the organization of said endothelial cells into vessels.
Angiogenesis plays a determining role in tumor growth and the development of metastases. In healthy tissues, an equilibrium exists between pro-angiogenic factors and anti-angiogenic factors; these factors being expressed or disregulated in tumor processes (Hanahan and Folkman, Cell, 1996, 86:353-364). Beyond a certain tumor volume, the growth of the tumor requires the development of a neovascularization which will bring it the necessary oxygen and nutrients. Tumor cells themselves secrete angiogenic factors and stimulate their microenvironment in order to increase the bioavailability of the factors necessary for the development of tumor angiogenesis.
The existence of a highly developed vascular network in tumors has been known for many years. As early as 1971, Folkman (N Engl J. Med., 1971, 285:1182-6) put forward the hypothesis that tumor growth was dependent on neovascularization (angiogenesis) and that the change from the latent phase to the aggressive phase was directly controlled by neovascularization, by means of diffusible substances originating from the tumor.
The control of angiogenesis involves several factors. It is triggered by a disequilibrium in the balance between the pro-angiogenic factors (for example, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), angiopoietins, or else angiogenin) and the anti-angiogenic factors (for example, endostatin and angiostatin, thrombospondins, vasostatin, prolactin, or else interferons).
Tumor cells, but also the inflammatory cells, macrophages, lymphocytes and myofibroblasts present in the tumor microenvironment, secrete angiogenic factors.
It is conventional to distinguish two phases during angiogenesis. The first phase is an induction phase which involves destabilization of the pre-existing tissue vascularization and destruction of the basal membrane surrounding the pre-existing vessels, and which requires the proliferation and migration of endothelial cells, and also the differentiation thereof to give capillary structures. The second phase is a stabilization/maturation phase during which perivascular cells (pericytes) are recruited, resulting in stabilization of the neocapillaries, and during which a basal membrane is reconstituted (Hanahan and Folkman, 1996, cited above).
It is now well established that the growth of a tumor and the formation of metastases are directly dependent on angiogenesis, suggesting that the inhibition of angiogenesis may represent an effective approach for preventing tumor progression and controlling metastatic diffusion.
Adrenomedullin (AM), isolated from human pheochromocytoma (cancer of the adrenal medulla), is a vasoactive peptide which acts locally as an autocrine/paracrine hormone and exerts multiple biological actions (Hinson et al., Endocr Rev., 2000, 21:138-67; Caron and Smithies, Proc Natl Acad Sci USA, 2001, 98:615-619; Shindo et al., Circulation, 2001, 104:1964-1971).
Several studies have shown that adrenomedullin binding sites are present in the cells of most tissues, such as the heart, the kidney, the brain, the lung and the adrenal gland. Binding sites are also present in tumor stromal cells. A role for adrenomedullin in tumor growth has also been demonstrated (Ouafik et al., Am J. Pathol., 2002, 160:1279-92; Martinez et al., J Natl Cancer Inst, 2002, 94:1226-37; Oehler et al., Oncogene, 2001, 20:2937-45; Ishikawa et al., Oncogene, 2003, 22:1238-1242).
This peptide may also play a positive role in the regulation of angiogenesis during vascular remodeling in response to ischemia, in the female reproductive system, during embryonic vascular development, and during the development and vascularization of the placenta.
Recently, several teams have demonstrated a role for adrenomedullin on endothelial cell proliferation, migration and invasion (Ouafik et al., 2002, cited above; Kim et al., FASEB J., 2003, 17:1937-9; Fernandez-Sauze et al., Int J Cancer, 2004, 108:797-804).
It has been shown, by means of in vivo and in vitro angiogenesis tests, that adrenomedullin acts on one of the last steps of neovascularization which consists of the reorganization of endothelial cells into tubules, independently of VEGF (Fernandez-Sauze et al., 2004, cited above).
Several studies demonstrate that adrenomedullin has angiogenic properties in most tumors (breast, prostate, colon, lung, kidney, respiratory tracts, bladder) (Ouafik et al., 2002, cited above; Fernandez-Sauze et al., 2004, cited above; Nikitenko et al., Br J Cancer, 2006, 94:1-7). In heterozygous adrenomedullin+/−mice, tumor volume decreases compared with the wild-type mice (Iimuro et al., Cir. Res., 2004, 95:415-423). This effect is associated with a reduction in neovascularization. Blocking the action of adrenomedullin with an antagonist (adrenomedullin2252) inhibits the growth of xenografted pancreatic tumors by destabilizing tumor vascularization (Ishikawa et al., Oncogene, 2003, 22:1238-1242). Similar effects have been observed in xenografts developed from glial tumor cells (Ouafik et al., 2002, cited above).
The discovery of the AMBP-1 (Adrenomedullin Binding Protein-1) serum protein suggests a regulation of the bioavailability of said adrenomedullin. AMBP-1 has been described and characterized as being human complement factor H. In general, binding proteins limit transport of the peptide in the interstitial space and access to its specific receptors. They also modulate the biological activity of the peptide and protect it against metabolic clearance by proteases, thus prolonging its half-life in the blood stream.
Adrenomedullin receptors (AMRs) are multiprotein complexes composed of the association of at least two proteins, CRLR (Calcitonin Receptor Like Receptor) and a RAMP protein (Receptor Actvity-Modifying Protein) (McLatchie et al., Nature, 1998, 6683:333-9).
The CRLR receptor was isolated in 1993 (Njuki et al., Clin Sci., 1993 4:385-8; Chang et al., Neuron., 1993, 6:1187-95). It comprises seven transmembrane G protein-coupled domains. The CRLR sequence was established in humans in 1996 (Aiyar et al., J Biol. Chem., 1996, 19:11325-9) and in pigs in 1998 Elshourbagy et al., Endocrinology, 1998, 4:1678-83). CRLR belongs to GPCR (G-protein-coupled receptor) class II, a class which groups together receptors for peptides such as glucagon and glucagon-like peptides (GLPs), secretin, parathormone or calcitonin. GPCRs are polypeptide in nature and comprise an extracellular portion carrying the ligand binding site, a seven-helix transmembrane portion and an intercellular portion in contact with the G proteins which provide the transfer and amplification of the signal received by the receptor. Three extracellular loops (called E1, E2 and E3) and three intracellular loops (I1, I2 and I3) can be observed (Bockaert and Pin, Embo J., 1999, 18:1723-1729). These proteins can be subject to post-translational modifications, such as N-glycosylation, or acetylation by lipid compounds sometimes forming a pseudo fourth intracellular loop (14) (Assie et al., EMC-Endocrinologie, 2004, 1:169-199).
For a few years, there was a certain amount of confusion regarding the exact nature of the adrenomedullin receptor owing to the homology of adrenomedullin with CGRP (Calcitonin Gene Related Peptide) and to it belonging to the calcitonin/CGRP/amylin peptide family. In 1998, McLatchie and collaborators (reference cited above) demonstrated that the CRLR receptor can generate two pharmacologically distinct receptors by association with a family of proteins, of 160 amino acids (14-21 Kda), with a single transmembrane domain, called RAMPs. CRLR is correctly functional only in the state of a dimer with a RAMP protein.
Three protein isoforms of RAMP exist: RAMP1, 2 and 3. These proteins have less than 30% sequence identity with one another, but have structural organization similarities. In humans, the genes encoding RAMP1, RAMP2 and RAMP3 are carried by chromosomes 2, 17 and 7, respectively. The RAMP proteins consist of a single transmembrane domain; the extracellular N-terminal end is relatively long and plays an important role in the specialization and the functionality of the receptor (CGRP or adrenomedullin) (Kuwasako et al., J Biol. Chem., 2001, 275:29602-9).
Two essential functions are attributed to the RAMP proteins: receptor determination and intracellular transport.                Receptor determination: the fundamental role of RAMP proteins is to define the specificity of the ligand which interacts directly at the cell surface. RAMP1 presents CRLR as a mature glycoprotein so as to form the CGRP receptor. Likewise, RAMP2 and RAMP3 present CRLR as a mature glycoprotein so as to form the adrenomedullin receptors. Thus, the nature of the RAMP proteins present in a cell type, the protein interactions which are established between the various partners (CRLR, RAMP1, RAMP2, RAMP3) and the proportion of each of the proteins allow the cells to respond specifically to various neuropeptides (Bühlmann et al., FEBS Lett., 2000, 486:320-4; Chakravarty et al., Br J. Pharmacol., 2000, 130:189-95).        Intracellular transport: CRLR requires the coexpression of the RAMP proteins for its transport to the cytoplasmic membrane (Sexton et al., Cell Signal, 2001, 13:73-83). It is also reciprocally the case: the RAMP proteins need CRLR for their translocation to the cell surface (Flahaut et al., J Biol. Chem., 2002, 277:14731-7).        
The growth of solid tumors is controlled by intratumor mechanisms and by interactions between the tumor and the surrounding tissue. In the quiescent phase, few vessels are detected. On the other hand, during the growth phase and during the invasive phase, there is an enormous amount of angiogenesis. A close correlation exists between tumor growth and the number of intratumor capillaries. Thus, angiogenesis-dependent solid tumors exhibit a latent pre-angiogenic phase and an aggressive angiogenic phase.
The treatment of tumors, in particular solid tumors, is based mainly on surgery, radiotherapy and chemotherapy. However, despite the progress obtained in these fields and the encouraging results, it proves to be essential to have new anticancer agents with a mechanism of action that is different from the available anticancer agents, inter alia in the field of targeted therapeutics, for increasing the efficacy of the treatment, in particular in the case of the appearance of resistance to a treatment and/or of adverse effects which are too great.
Alongside therapies aimed at the destruction of proliferative cells (chemotherapy) and hormone therapy in the context of hormone-dependent cancers (breast, prostate), targeted therapeutics are aimed at all the pathways which contribute to tumor development, such as proliferation signals, the cell cycle, apoptosis, invasion or angiogenesis (Folkman, Nat Rev Drug Discov., 2007, 6:273-286; Neri and Bicknell, Nat Rev Cancer, 2005, 5:436-446).
The presence of tumor neoangiogenesis associated with overexpression of the mRNAs of angiogenic factors such as VEGF and FGF-2 has resulted in the development of inhibitors (specific antibodies, antisense oligonucleotides, pharmacological inhibitors) by several pharmaceutical companies. A certain number of molecules, currently undergoing clinical trials, are involved in therapeutic protocols jointly with conventional treatments.
However, it is known that the use of anti-VEGF antibodies for treating tumors, even if it gives good results (rapid arrest of tumor growth), has considerable toxic effects. In addition, a recurrence (resumption of tumor growth) can occur in the more or less short term after treatment has stopped. Furthermore, these treatments are aimed at the endothelial cells, but not all the cells of the tumor stroma that are involved in establishing a functional neoangiogenesis.
There still remains therefore a need for effective treatments aimed at blocking, or even inhibiting, tumour growth and/or at regressing tumor size.
In this context, the targeting of adrenomedullin via its receptors for therapeutic purposes constitutes a relevant approach owing to its mechanism of action which relates to the endothelial cells, but also, unlike VEGF, to the tumor cell and to all the cells of the stroma, particularly the pericytes.
Thus, Fernandez-Sauze et al. (2004, cited above) have shown that, in vitro, mixtures of anti-CRLR/anti-RAMP2 polyclonal antibodies, on the one hand, and anti-CRLR/anti-RAMP3 polyclonal antibodies, on the other hand, inhibit the specific binding of adrenomedullin to its receptor on several cell types and block the formation of vascular tubes. More recently, it has been described, in International Application WO 2007/045927, that pharmaceutical compositions comprising antibodies which bind specifically to the RAMP2 or RAMP3 human proteins can be of use for treating or preventing cancer, via, for example, the inhibition of angiogenesis or of the proliferation of cancer cells.
The inventors have prepared antibodies which bind to the proteins that form adrenomedullin receptors and have shown, surprisingly, that a mixture of at least three antibodies which bind to three different proteins forming adrenomedullin receptors, more particularly the hCRLR, hRAMP2 and hRAMP3 proteins, exhibit an antitumor efficacy in vitro and/or in vivo which is significantly greater compared with the use of a single anti-CRLR, anti-RAMP2 or anti-RAMP3 antibody, or even compared with the use of a mixture of two anti-CRLR/anti-RAMP2 or anti-CRLR/anti-RAMP3 antibodies.
The subject of the present invention is therefore a mixture of at least three antibodies and/or fragments of said antibodies which bind to three of the proteins forming adrenomedullin receptors, each antibody and/or antibody fragment binding to a different protein, for use as a medicament.